Cell surface display refers to a technology of expressing proteins or peptides in a form fused with a suitable surface anchoring motif on the surface of cells, such as gram negative and positive bacteria, molds, yeasts and animal cells (Lee, S. Y. et al., Trend. Biotechnol., 21:4552, 2003). In 1980s, the surface-expression system was first developed by expressing peptides or small proteins fused with pIII of a filamentous phage with a relatively simple surface. Although the cell surface display using the phage was used in the screening of antibodies, epitopes, high-affinity ligands and the like, the size of proteins which can be expressed on the phage surface is relatively limited. Thus, as an alternative substitute therefor, a cell surface expression method for stably expressing foreign proteins on the surface of microorganisms using a surface protein of microorganisms, such as bacteria or yeasts, as a surface anchoring motif, has been developed.
In order to express a foreign protein on the surface of cells using the outer membrane protein of a certain organism, a suitable surface protein and the foreign protein should be linked with each other at a gene level to biosynthesize a fusion protein, which should be stably passed through a cell inner membrane and attached, and then maintained on the cell surface. For this purpose, a protein having the following properties is preferably selected for use as a matrix for surface expression. Namely, (1) it has a secretion signal capable of passing through the cell inner membrane, at the N-terminal end; (2) it must have a targeting signal which can be stably attached on the surface of a cell outer membrane; (3) it can be expressed on the cell surface in large amounts within range of having no adverse effect on the growth of cells, such that the protein can show high activity; and (4) it is stably expressed regardless of its size such that it should be able to be used in various reactions (Georgiou et al., Trend. Biotechnol., 11:6, 1993). Such a matrix for surface expression needs to be genetically engineered such that it is inserted into the N-terminal end, C-terminal end or central portion of the outer membrane protein on the surface of the host cells (Lee et al., Trend. Biotechnol., 21:45-52, 2003).
In order to successfully express target peptide or protein on the surface of bacteria, such as E. coli with a complex membrane structure, it is first required to select a surface anchoring motif by which foreign proteins to be expressed on the cell surface can be stably and efficiently transported to the cell surface. Surface anchoring motifs which have been used in E. coli till now include outer membrane proteins, lipoproteins, autotranspoters, the subunits of surface appendages, and S-layer proteins. Among them, the outer membrane proteins have several advantages such as efficient secretory signal, unique membrane spanning structures providing fusion sites, and strong anchoring structures. Owing to such advantages, the outer membrane proteins have been frequently used as a surface anchoring motif.
Outer membrane proteins such as OmpA, OmpS, LamB, OprF, PhoE and the like have widely been used for the expression of peptides, antibodies, domains, receptors, which have a relatively small molecular weight (Agterberg, M. et al., Gene, 88:37, 1990; Lang, H. et al., Eur. J. Bacteriol., 267:163, 2000). Since the C-terminal and N-terminal ends of inserted foreign proteins must be placed close to each other in three dimensions, these cell surface display system show low stability for the large protein. In fact, if LamB or PhoE is inserted with a foreign protein comprised of 50-60 or more amino acids, it will encounter limitations in its structure, which make it impossible to form a stable membrane protein. Also, E. coli porin (outer membrane protein) was used only in epitopes or metal binding motifs other than proteins comprised of at most 150 amino acids (Stahl, S. et al., Trends Biotechnol., 15:185, 1997; Kjaergaad, K. et al., Appl. Environ. Microbiol., 66:10, 2000).
Cell surface display using a bacterial secretion system is used in a very wide range of applications. Also, it can be used in various applications depending on proteins or peptides which are expressed on the cell surface. The expression of a certain protein on the cell surface allows simple screening of peptides, antibodies or receptors (Francisco, J. A. R. et al., PNAS, 91:10444, 1993), and the expression of antigen epitopes on the cell surface allows the production of live vaccines which can show a powerful immune response. Also, a certain enzyme required in fine chemicals, agricultural chemicals or medicines, which have been expressed on the cell surface, may be used as a whole-cell biocatalyst, or proteins capable of degrading contaminants or adsorbing metal ions, which have been expressed on the cell surface, may be used in bioremediation (Charbit, A. et al., Gene, 70:181, 1988; Sousa, C. et al., J. Bacteriol., 180:2280, 1998; Richins, R. et al., Nat. Biotechnol., 15: 984, 1997).
Various kinds of surface anchoring motifs have been reported till now. However, targets which can be expressed on the cell surface by one surface anchoring motif are limited, so that the development of surface anchoring motifs which is different from each other, is needed to express various proteins on the cell surface.
FadL from E. coli which is a protein associated with the fatty acid metabolism of E. coli is involved in fatty acid transport. Regarding the membrane topology of the FadL protein, the N-terminal and C-terminal ends of the protein are found in the peripheral cytoplasm, 10 loops are out of the outer cell membrane, and 9 loops are out towards the peripheral cytoplasm (Cristalli et al., Arch. Biocheem. Biophys., 377: 324, 2000).
Since FadL is a protein of E. coli itself, it can easily express foreign proteins in a form fused with the foreign protein using E. coli as a host cell. Also, this protein has 10 external, loops providing various points which can be fused, thus, it is expected that the FadL protein can increase the possibility of cell surface expression of foreign proteins. Furthermore, the FadL protein has a stability that has been pointed out as a shortcoming of enzymes expressed on the cell surface in several surface expression systems. In other words, this protein has an advantage in that it allows the enzymes expressed on the cell surface to maintain their activity for a long time period under various conditions (high temperature, high pH, organic solvent, etc.).
Accordingly, the present inventors have truncated a gene following the ninth loop expected to have the highest expression possibility of 10 loops which are out of the outer cell membrane in the outer membrane protein, fused a foreign protein (lipase) to the truncated loop position of E. coli, and expressed the lipase on the surface of cells. As a result, the present inventors confirmed that the lipase expressed on the cell surface is useful in bioconversion reactions, thus perfecting the present invention.